Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Switzerland), was placed inside the chromatographic oven to<br />
bypass the combustion furnace when required. The valve has an<br />
extension between the body and the actuator which allows the<br />
valve to be mounted in the heated zone while the handle remains<br />
outside at ambient temperature. This valve prevented the solvent<br />
from entering the combustion unit and therefore enlarged the<br />
catalytic activity of the Cu and Pt wires. Additionally, the valve<br />
allowed the direct connection of the column with the EI source<br />
for conventional GC-MS work in order to identify the different<br />
species under analysis by their fragmentation pattern. Moreover,<br />
such qualitative analysis is essential to assess peak purity for each<br />
compound. For postcolumn isotope dilution analysis the valve was<br />
initially in the “load” position (combustion oven bypassed) and<br />
the valve was manually switched to the “inject” position at the<br />
same time that the EI filament switched on. All connections<br />
between the valve and the rest of the components of the system<br />
were performed by means of 0.32 mm i.d. deactivated fused silica<br />
capillaries and fixed to the valve with appropriate polyimide coated<br />
fused silica adapters (VICI AG International, Schenkon, Switzerland).<br />
Combustion Furnace. The laboratory-made combustion furnace<br />
consisted of a 60 cm long ceramic tube (3 mm O.D., 0.5 mm I.D.)<br />
(Elemental microanalysis, Devon, U. K.) filled with copper and<br />
platinum wires and heated by a Nichrome wire. Proper thermal<br />
isolation of the combustion furnace was provided with glass wool.<br />
The combustion furnace was set vertically on top of the GC with<br />
the lower end of the ceramic tube inside the chromatographic<br />
oven to avoid cold spots after the separation. High temperatures<br />
were accurately controlled using a temperature sensor and an<br />
external controller, allowing temperature settings inside the tube<br />
to be within ±1 °C over a wide temperature range (50-1200 °C).<br />
The copper wires were previously oxidized by passing an oxygen<br />
flow (1 mL/min) at 450 °C during 4-5 h and this procedure was<br />
performed on a weekly basis to preserve its oxidizing capabilities.<br />
Fused silica capillaries (0,32 mm i.d.) were used to connect the<br />
furnace to the valve and to the ion source respectively. For this<br />
purpose a length (∼1 cm) at one end of each capillary was<br />
uncoated, to prevent the polyimide coating to be burnt, and<br />
introduced into the ceramic tube. Reducing unions 1/8′′ to 1/16′′<br />
(SGE, Victoria, Australia) and appropriate graphite ferrules (0.5<br />
mm i.d.) were used to hold the capillaries in place at both sides<br />
of the ceramic tube. For oxidation and combustion, the furnace<br />
was operated at 850 °C.<br />
Gas Cylinder and Mass Flow Controller. The 13 CO2 container<br />
was a dual inlet 5 L high pressure stainless steel gas cylinder<br />
(Iberfluid, Barcelona, Spain). The cylinder was equipped on<br />
one end with, in this order, an opening valve, a Swagelok tee<br />
where helium could be introduced from a high pressure<br />
cylinder, and a second opening valve connected to the other<br />
end of the tee. This second valve was connected to a septum<br />
for the manual injection of gases into the cylinder. Before the<br />
cylinder was pressurized, 13 CO2 was injected into the container<br />
by means of a gastight syringe (Hamilton, Reno, U. S. A.)<br />
through this valve. After the injection of the tracer, the valve<br />
was closed and the container was pressurized up to 6 bar with<br />
helium using the connection in the Swagelok tee. When the<br />
set pressure was reached the filling valve was also closed. At<br />
the other end of the cylinder a pressure gauge, an opening<br />
6864 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
valve and a mass-flow controller (Bronkhorst, Ruurlo, Netherlands)<br />
calibrated for He were coupled for the accurate control<br />
of the tracer flow. The opening valve was closed during the<br />
filling of the cylinder, remaining open the rest of the time. The<br />
flow rate for the postcolumn spike was set at 0.5 mL/min.<br />
Effluent and spike flows were mixed after combustion by means<br />
of a 0.25 mm bore stainless steel microvolume “Y” connector<br />
(VICI AG International, Schenkon, Switzerland).<br />
The whole instrumental setup is shown in Figure 1. As can be<br />
observed, the instrument can be operated in the standard GC-<br />
MS configuration (qualitative) or in the combustion-postcolumn<br />
configuration (quantitative) depending on the position of the<br />
switching valve.<br />
Procedures. Preparation of 13 CO2. The spike was prepared<br />
from 13 C enriched Na2CO3 (99%). An accurate weighed amount<br />
(∼200 mg) was placed in a 25 mL three-necked round-bottomed<br />
flask, previously purged with He to avoid natural abundances<br />
CO2 contamination from ambient air. A small quantity (300 µL)<br />
of concentrated H3PO4, was injected into the flask through a<br />
septum cap. After the acid-base reaction, 4 mL of the gaseous<br />
phase, containing 13 CO2 diluted in He, were removed using a<br />
gastight syringe and injected into the container shown in<br />
Figure 1.<br />
Calibration of the 13 CO2 Postcolumn Flow. The flow rate of<br />
the spike could be accurately controlled by the mass flow<br />
controller between 0.1 and 5 mL min -1 . In our experiments, it<br />
was set at 0.5 mL min -1 . The exact mass flow (ng of 13 CO2 per<br />
min) being mixed with the natural CO2 coming from the<br />
column and combustion furnace was determined by adding<br />
internal standards of known concentration spiked to the sample<br />
as described before. 11,15<br />
Quantification using Postcolumn Isotope Dilution. In our case,<br />
the isotope ratio 12 C/ 13 C was measured as the signal ratio at<br />
masses 44 and 45 (I44/I45) corresponding to the continuous<br />
blend of natural abundance 12 CO2 present in the chromatographic<br />
eluent and the enriched 13 CO2, added postcolumn.<br />
Selected Ion Monitoring (SIM) at masses 44.0 and 45.0 was<br />
performed for the duration of the chromatogram with 70 ms<br />
integration time per mass. The mass window was ∼0.1 mass<br />
units. Then, the isotope ratio in the blend, Rb ) I44/I45, was<br />
calculated to build the isotope ratio chromatogram (Rb vs time).<br />
The postcolumn isotope dilution equation, 7 shown as equation 1<br />
below, was then applied to every point in the chromatogram<br />
to obtain the mass-flow chromatogram (ng of C/min vs time).<br />
The integration of the mass flow chromatogram directly<br />
provided the amount of carbon (in ng) eluted in each chromatographic<br />
peak.<br />
MF n ) MF t<br />
AW n<br />
13<br />
At AWt An 12( Rb - Rt 1 - RbRn) In this equation MFn corresponds to the mass flow of carbon<br />
from the natural abundance sample injected, whereas MFt<br />
corresponds to the mass flow of carbon from the postcolumn<br />
spike or tracer. AWn and AWt correspond to the atomic weight<br />
of carbon in the sample and tracer, respectively. The isotope<br />
abundances At 13 and An 12 correspond to the isotopic composition<br />
of 13 C in the tracer and 12 C in the sample. Finally, Rt is the<br />
(1)